Particle analysis in an acoustic cytometer

ABSTRACT

The present invention is a method and apparatus for acoustically manipulating one or more particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/571,629, entitled “Particle Analysis in an Acoustic Cytometer,” filedAug. 10, 2012, which is a continuation of U.S. patent application Ser.No. 12/283,461, entitled “Particle Analysis in an Acoustic Cytometer,”filed Sep. 11, 2008, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/008,422, entitled “Particle Analysis in anAcoustic Cytometer,” filed Dec. 19, 2007. The foregoing applications areincorporated herein by reference in their entireties for any and allpurposes.

TECHNICAL FIELD

Embodiments of the present invention relate to acoustic cytometry andmore specifically to particle analysis in an acoustic cytometer.

BACKGROUND

It was realized early in the development of flow cytometry that theangular dependence of the light scattering intensity from a particlecontains a wealth of information related to a particle's intrinsic andextrinsic properties. For example, Mullaney, et al. experimentallydemonstrated the use of forward light scatter (0.5-2 degrees) for cellsize estimation. In the same time period, it was also realized that cellorientation relative to the incident light beam can introduce artifactsthat affect population resolution and instrument sensitivity forparticles that do not possess axial symmetry parallel to the flowdirection. Loken et al. showed that nonspherical particles in the formof fixed chicken erythrocytes yield a bimodal scattering distributionthat is related to rim-on or face-on illumination of the disk-cellstructure. Particle orientation dependent scattering effects due toasymmetries that are apparent in the complex geometrical shape of spermcells have also been demonstrated. Several researchers have suggestedpassive solutions to orienting particles by shaping the sample nozzle tointroduce asymmetries into the velocity flow field of the hydrodynamicsheath system. It has been shown that the most critical aspect inefficient X and Y sperm discrimination in flow cytometric sorting is theorientation of the sperm in the optical scattering plane. Recently,novel nozzle geometries have demonstrated proper orientation of up 60%of the sperm heads in the optical scattering plane at analysis ratesnear 2000 particles/s dramatically affecting the sorting efficiency.Other researchers have addressed particle asymmetries by collecting dataover a large array of scattering angles using scanning flow cytometry(SFC) at the cost of lower particle analysis rates (approximately 400particles/s). Systems have demonstrated continuous angular scatteringdata spanning 70 degrees but the data is taken relative to an arbitraryparticle orientation that results in large variations of the collectedangular spectra for asymmetric particles.

One of the earliest large-scale demonstrations of separating biologicalcells using acoustic forces was done by Doblhoff, et al. In that system,acoustic radiation pressure was used for selective retention of viablehybridoma cells for the purpose of removing nonviable cells and smallercellular debris from a 20 liter bioreactor. That system was based on amulti-planar transducer design and demonstrated viable cell retentionrates as high as 99.5% with varying results for cellular debrisrejection. That early system required high power input (in excess of15W) thus necessitating a cooling unit for the drive transducers. Morerecently, Feke and coworkers developed a novel particle separationstrategy that relies on both acoustic radiation pressure and secondaryacoustic forces. A high-porosity polyester mesh (pore size two orders ofmagnitude greater than particle size) in an acoustic standing waveserved as a collection matrix whereby particles at nodal locations weretrapped within the mesh and secondary acoustic forces formed particleagglomerates and created an attractive force at the mesh surface. In asimilar demonstration of retention of hybridoma cells, retentionefficiencies of about 95% were achieved with negligible effects on cellviability. This system achieved high cell densities of approximately1.5×10⁸ cells/mL with only hundreds of milliWatts of input power.

SUMMARY

An embodiment of the present invention comprises an apparatus thatacoustically manipulates a particle and stops flow of the particle. Thisapparatus preferably comprises a capillary for flowing a fluidcontaining the particle therein, an acoustic signal producing transduceracoustically manipulating the particle, and a stop flow device. The stopflow device is preferably a pump or one or more valves. This embodimentcan comprise a particle sorter, a particle fractionator, and/or a flowcytometer. This embodiment can further comprise an analyzer to analyzethe particle and/or an imager.

Another embodiment of the present invention comprises an apparatus thatacoustically manipulates a particle and reverses flow of the particle.This apparatus preferably includes a capillary for flowing a fluidcontaining the particle therein, an acoustic signal producing transduceracoustically manipulating the particle, and a reverse flow device. Thereverse flow device preferably comprises a pump and/or one or morevalves. The apparatus of this embodiment can further comprise ananalyzer for analyzing the particle and/or an imager. The apparatus ofthis embodiment can optionally comprise a sorter, a fractionator, and/ora flow cytometer.

Yet another embodiment of the present invention comprises an apparatusthat acoustically aligns and orients a particle in a flow stream. Thisapparatus preferably comprises a capillary for flowing a fluidcontaining the particle therein, an acoustic signal producing transduceracoustically manipulating, aligning and orienting the particle, and aparticle analyzer. The apparatus of this embodiment preferably comprisea flow cytometer, a particle fractionator, and/or a particle sorterwhere the sorter sorts the particle based on size. The apparatus canoptionally include an imager. The acoustic signal producing transducerof this embodiment preferably aligns the particles in either a polardirection about a flow axis or aligns the particle in a direction offlow. The particle of this embodiment can be a red blood cell, aplatelet or a sperm.

One embodiment of the present invention comprises an apparatus thatanalyzes a particle. The apparatus of this embodiment preferablycomprises a capillary for flowing a fluid containing the particletherein, a radial acoustic signal producing transducer that acousticallyorients non-axial symmetric particles in said capillary, a transportdevice that transports said particles through an interrogation point,and a particle analyzer. In this embodiment, the radial acoustic signalproducing transducer preferably aligns the particle in the capillary andconcentrates the particle in the capillary. The radial acoustic signalproducing transducer can also create an acoustic field that aligns theparticle. This embodiment can also optionally include a hydrodynamicsheath that aligns the particle. The apparatus of this embodiment canfurther comprise an imager.

Another embodiment of the present invention comprises an apparatus thatanalyzes a particle in a fluid. The apparatus of this embodimentpreferably comprises a capillary for flowing a fluid containing theparticle therein, an acoustic signal producing transducer thatacoustically maintains particle focus in said capillary regardless offlow rate, and a particle analyzer for analyzing the particle. Thisapparatus can further comprise a stop flow device and/or a reverse flowdevice. The apparatus of this embodiment also preferably comprises animager.

Still another embodiment of the present invention comprises an apparatusthat sorts particles by size. This apparatus preferably comprises acapillary for flowing a fluid containing the particles therein, and aradial acoustic signal producing transducer acoustically sorting andseparating the particles in said capillary by size. This embodiment canfurther comprise a flow cytometer and/or a particle analyzer and/or animager. The apparatus also preferably comprises a particle sorter.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIG. 1 is an embodiment of the present invention illustrating a linedrive capillary where particles are acoustically focused to the centralaxis of the capillary;

FIG. 2A is a density plot of acoustic force potential in the crosssection of a circular capillary driven by a line source according to oneembodiment of the present invention;

FIG. 2B illustrates an induced particle rotation to lie in a stableforce equilibrium plane according to one embodiment of the presentinvention;

FIG. 3 is an embodiment of the present invention illustrating a linedriven acoustic fractionator where large particles are transported tothe capillary axis while smaller particles remain unaffected by theacoustic field;

FIGS. 4A and 4B illustrate particles flowing through a capillary in arandom orientation when the acoustic field is off and then particlesaligned coincident with the capillary axis upon excitation of theacoustic field according to one embodiment of the present invention;

FIGS. 5A-5C illustrate selective fractionation of particles that areapproximately 1 pm and approximately 10 pm in a line drive capillaryaccording to one embodiment of the present invention;

FIG. 6 is an embodiment of the present invention illustrating a sampleinput into a flow cytometer where the sample is concentrated reducingits volume and thus reducing the analysis time in flow cytometryapplications.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As used herein “a” means one or more.

As used herein “capillary” means a flow channel or chamber having ashape selected from rectangular, elliptical, oblate circular, round,octagonal, heptagonal, hexagonal, pentagonal, and triagonal.

In one embodiment of the present invention, acoustic radiation pressurepreferably focuses particles to the center of a capillary. Thisembodiment enables fundamental changes in the way single cells orparticles can be either analyzed or processed in a flow stream. Thissample delivery embodiment expands the analysis capabilities of flowcytometry by improved analysis and/or preanalysis sample preparation andpurification.

A non-limiting embodiment of an acoustic particle focusing device isillustrated in FIG. 1. This device preferably comprises acousticallydriven capillary 10 where an acoustic particle trap oriented parallel tothe flow direction comprises line source 12 and fluid 18 withparticles/cells 16. This embodiment enables removal of high-velocitysheath flow associated with hydrodynamic focusing and allows extendedparticle transit times within the optical interrogation region whilemaintaining a high particle analysis rate due to an inherent in-lineparticle concentration effect. Additionally acoustic focusing of theparticle stream provides the ability to stop and reverse the flowdirection without defocusing the particle stream while maintainingparticle registration. The increased particle transit times provide anopportunity for high-sensitivity optical measurements that use low-powerlight sources and less sensitive optical components. Control of flowdirection and rate allows for reanalysis of targets of high significancethereby minimizing uncertainties in the scattering data associated withsystem level fluctuations.

An additional property of an acoustically driven flow chamber is anon-axisymmetric force field that is created within the flow stream.Force asymmetries within the flow chamber orient nonspherical cells orparticles such that consistent scattering signatures, that are notpossible with standard hydrodynamic flow systems, are produced bypositioning asymmetric particles in a predetermined orientation withinthe optical scattering plane. In this embodiment, optical scatteringdata associated with specific particle orientation can, for example,distinguish between different types of bacteria based upon particleasymmetries and improve analysis and sorting of irregular cell typessuch as RBC's and sperm cells. The asymmetric force field also yieldsrepeatable orientations of particle clusters that are formed from thecoalescence of multiple microspheres or cells (e.g. ‘dumbbell’ shapesfrom agglutination of two particles). Discrimination of particleclusters can be made easier by pulse shape analysis and angularscattering interpretation due to the unique induced orientation of thecluster relative to the scattering plane (e.g. central axis of the‘dumbbell’ always parallel to the flow axis).

An acoustically line-driven capillary embodiment of the presentinvention brings new modes of particle and cell analysis to thedetection system of a flow cytometer, and is also employed in particleor cell separations for in-line sample preparation. A primary advantageof acoustic radiation pressure is that it can be used in fairly largechambers with high volume throughput. The acoustic field acts similarlyon most biological particles and is thus inherently nonspecific wherebymost biological particles are transported to the same spatial location.The magnitude of the field, however, is highly size dependant and makesacoustic radiation pressure an excellent candidate in applications thatrequire high throughput, pre-analysis in-line separating of particlesbased upon size e.g. sperm from vaginal cells in forensic analysis,virus from bacteria or intact cells from cellular debris. In thedescription above, a line-driven capillary with a cylindrical geometryis used as the acoustic focusing device, but general geometry (square,rectangular, elliptical, oblate circular, etc.) devices that employacoustic radiation pressure to position the particles can be used inapplications of particle separation, debris rejection, particlealignment, and sample purification.

Acoustic Radiation Pressure

The force on a particle resulting from acoustic radiation pressuredepends upon frequency of excitation, pressure amplitude within themedium, and the density/compressibility contrast between the particleand the host medium. Within an acoustic standing wave, it is atime-averaged drift force which transports the particles to a nodal oran anti-nodal position. The expression for the acoustic radiation forcepotential U on a spherical particle in an acoustic standing wave isgiven by:

$U = {\frac{4}{3}\pi \; {a^{3}\left\lbrack {{\left( {\beta_{o}\frac{\langle p^{2}\rangle}{2}} \right)f_{1}} - {\frac{3}{2}\left( \frac{\rho_{o}{\langle v^{2}\rangle}}{2} \right)f_{2}}} \right\rbrack}}$

Here, a is the particle radius, 130 is the compressibility of thesurrounding fluid, and pc, is the density of the surrounding fluid. Thepressure and velocity of the acoustic field in the absence of theparticle are described by p and v, respectively, and the bracketscorrespond to a time-averaged quantity. The terms f1 and f2 are thecontrast terms that determine how the mechanical properties of theparticle differ from the background medium. They are given by:

$f_{1} = {1 - \frac{\beta_{p}}{\beta_{o}}}$$f_{2} = \frac{2\left( {\rho_{p} - \rho_{o}} \right)}{2\left( {\rho_{p} - \rho_{o}} \right)}$

The subscript _(p) corresponds to intrinsic properties of the particle.The force F acting on a particle is related to the gradient of the forcepotential by:

F=−vU

Particles are preferably localized at positions where the potential Udisplays a minimum. (For a circular cross section capillary, a potentialminimum is coincident with the axis of the capillary forming theparticle trap in FIG. 1 when driven in a dipole type mode. Other modesexist and are advantageous for spatial positioning of particles inlocations other than the axis of the capillary for selectedapplications.)

Acoustic Line-Driven Capillary

Forces resulting from acoustic radiation pressure are preferably aneffective means to localize particles in an arrangement similar tohydrodynamic focusing without the need for sheath fluids. Theline-driven capillary of the present invention has been proven effectivein sheath replacement. (A capillary with a source aperture larger than aline contact can yield similar results. This embodiment has demonstratedacoustically driven capillaries with source apertures that have anextended contact length along the circumference of the capillary thatspans more than about 45 degrees.) It is constructed from a capillarythat is driven by a piezoceramic source in contact with its outer wall.Vibration of the structure creates a localized pressure node along thecentral axis where an axial particle trap is formed. A diagram of thisdevice is given in FIG. 1. Particles in a dilute suspension enter thedevice from the top and experience a radial force that transports themto the pressure node as they flow through the system. In an embodimentof the present invention, the particles contained in a sample aresimultaneously concentrated and aligned in single file as they are thentransported through the interrogation laser. The particles aretransported through the interrogation laser via various transportdevices, including but not limited to, a pump and/or one or more valves.

Implementation of acoustic particle focusing preferably allows new flowcytometry techniques and methods to evolve due to fundamental changes inthe way particles are positioned within the sample cell. Concentric flowstreams with different flow velocities are not required as withconventional hydrodynamically sheath-focused systems. Acousticallyfocused sample streams can be stopped, slowed, reversed or anycombination thereof without degrading alignment of the particle streamwithin the flow chamber. The increased residence time within theacoustic field produces a stream of particles whose focus is actuallyimproved. Additionally, the flow can be reversed with no adverse effecton particle alignment within the flow chamber allowing rare targets tobe repeat analyzed or stopped for extended analysis such as spectraldecomposition of the scattered/fluorescence signature.

One of the unique flow capabilities of the present invention is theability to select the sample delivery rate. By slowing cell/particletransit times (approximately 20-100 times slower than conventionalsystems) higher sensitivity optical measurements and measurements ofphotonic events that require longer interrogation times such asluminescence are possible.

Particle Orientation in a Standing Acoustic Wave Field

A known orientation of a particle as it passes through the interrogationregion enables light scatter/fluorescence measurements that providesignificant insight into cellular structure and intrinsic opticalproperties. The removal of several degrees of rotational freedom provesan invaluable tool to flow cytometry by increasing the value of currentlight scatter measurements by calibrating them to a specific orientationof a cell/particle and allowing reasonable consideration of new scatterangles as measured parameters. The acoustic line-driven capillary of thepresent invention (or other methods to introduce acoustic radiationpressure into the flow cell) is an active means to rotate and alignparticles in both the direction of flow and in polar directions aboutthe flow axis to yield angular calibrated scattering data for particlesthat are non-spherical. The force experienced by a particle in anacoustically driven tube is inherently non-axisymmetric within the crosssection of the flow plane. The acoustic force distribution is dipolar innature for particle focusing to the tube axis yielding force reflectionsymmetries within the plane. Calculations of the acoustic forcepotential U for a particle in a line driven tube in a dipole type modeas one example of this method are shown in FIG. 2(a) where the acousticforce F_(u) can be obtained by

F=−vU

where the flow direction is into the page. The in-plane force potentialpossesses reflection symmetry about two planes that intersect thecentral axis. The first plane of symmetry intersects central axis 20 andline drive 22 and the second plane of symmetry lies perpendicular to thefirst. Though two symmetry planes exist within the displayedtwo-dimensional acoustic force potential, only one results in stableequilibrium 24 location with respect to particle rotation. All regularparticles will rapidly rotate into stable equilibrium 24 under smallperturbations within the flow field as shown in FIG. 2(b).

Incorporating a third dimension into the force field calculation (axialcomponent) yields an additional restriction in the rotational freedom ofa particle induced by the acoustic force field. Calculations show thatrod shaped particles (particles with two equal minor axes and one majoraxis) will align their major axis with the axis of the capillary.Particles with bilateral symmetry, e.g. red blood cells, will align onemajor axis parallel to the flow axis and the other major axis paralleltb the stable symmetry plane denoted by the white dotted line in FIG.2(a).

Acoustic Separation of Cells and Cellular Debris

For particle transport to occur in an acoustically driven chamberaccording to one embodiment of the present invention, the acoustic forcemust be large enough to overcome the Brownian motion of the particleswithin the suspending medium. The magnitude of the acoustic radiationpressure force experienced by a particle is directly proportional to theparticle volume, drive level of the acoustic field, mechanicalproperties of the medium and particle, and the spatial gradient of theacoustic field. For this reason, (due to a cubic relationship ofparticle radius) larger particles can be transported in an acousticfield at lower pressure amplitudes and excitation frequencies (smallergradients) than smaller particles. (This is also true for particles thathave a greater relative difference in their mechanical propertiesrelative to the background medium.)

One aspect of one embodiment of an acoustic separation system of thepresent invention is that it can operate clog-free (no filter) withalmost zero pressure drop across the unit. Due to the size dependenceinherent in the acoustic radiation force and thermal particle motion, anembodiment of the present invention can separate samples at the frontend of a flow stream based upon particle size and mechanical contrast.Acoustic forces are used to purify samples by concentrating analytes ofinterest at a specified location for selective collection leavingbackground debris unaffected. Such a system reduces analysis time forsamples with high particulate backgrounds on a flow cytometer by greatlyreducing the particle count and increasing data quality. For instance,Bossuyt showed that cellular debris within whole blood samples preparedby selected lysis methods can yield scattering events that account forup to 80% of all events in CD45 cell counting on a flow cytometer. Maceynoted that certain whole blood lysis methods for preparing lymphocytesfor flow cytometry analysis can result in poor forward and side scatterresolution due to the presence of residual cell fragments. In oneembodiment of the present invention, an in-line purification device,such as a line-driven capillary located just prior to the sample inletof a flow cytometer as shown in FIG. 3, is used to transport largeparticles of interest 30 (e.g. lymphocytes) to central axis 32 of thesample stream while smaller particles 34 such as cellular debris andproteins contained within the lysate remain unaffected. This isespecially true for cellular debris with less mechanical contrast thanthe particles of interest. The central core of the sample stream is thenfed into the flow cytometer and the remaining lysate is discardedeliminating a large particulate concentration from the sample. It shouldbe noted that this method of sample preparation can be used as a samplepurification step for any type of particle/cellular analysis where thereduction of background particulate count is advantageous

Particle Orientation in a Standing Acoustic Wave Field

Example 1

To demonstrate the effects of the acoustic field to induce deterministicparticle orientation, experiments using particles with aspect ratiosgreater than unity were conducted with a line driven capillary. In oneexample, the capillary was made of glass and had an by inner diameter ofapproximately 500 pm and an outer diameter of approximately 1000 pm. Anacoustic source was attached to the outer surface of the capillary(parallel with the axis of the capillary) and operated at approximately1.78 MHz and approximately 10 v_(pp). A suspension of circularcylindrical carbon fibers in deionized water was transported down thetube with a syringe pump. The particles were then imaged through amicroscope. The fibers had a minor axial dimension of approximately 8 pmwith varying greater major axis dimensions.

FIG. 4A illustrates the sample as it flowed through the capillary (flowis from left to right). Fibers were seen in random orientations as theywere entrained in the fluid and transported through the system when noacoustic field was present. Upon acoustic excitation of the capillary,the fibers were transported and rotated to align coincident and parallelwith the axis of the capillary, see FIG. 4B. The alignment shown herewas due to the acoustic radiation pressure force aligning the major axisof the particles along the axis of the capillary.

Field-Based Particle Size Selection for In-Line Sample Purification andSeparation/Concentration

By varying the drive voltage and/or the frequency of excitation of theacoustic source in an acoustically driven capillary, a binaryfractionation of particles by size can be achieved. This effect is aresult of the reduced acoustic force felt by the smaller particles dueto the cubic dependence of the acoustic force on particle radius. inapplication, the larger particles contained within the central core ofthe capillary are fed into a smaller, coaxial capillary discarding theconcentric flow field containing small particulates. The purified samplecan be taken for further sample preparation steps or fed real-time intoa flow cytometer or other means of analysis. Depending upon theapplication, the fluid outside the central core may also be considered avaluable sample to be collected and used for analysis.

Example 2

Results from preliminary experiments demonstrating the size selectioncapability as a function of drive level are illustrated in FIGS. 5A-5C.In this example, an acoustically driven capillary was oscillated atapproximately 1.78 MHz. A suspension of latex microspheres containingapproximately 1 μm diameter fluorescent spheres and approximately 10 μmdiameter non-fluorescent spheres are pumped through the drivencapillary. The volume fraction of particles was approximately 2.5×10⁻⁵.The capillary is defined by an inner diameter of approximately 500 pmand outer diameter of approximately 1000 μm.

FIG. 5A is a photograph taken through a fluorescence microscope wherethe approximately 10 pm particles are viewed as large circularinclusions and the approximately 1 μm particles are viewed as a grainybackground. (The fluorescence signal from the approximately 1 μmparticles is too low to be observed under the operating conditions ofthe experiment.) Under low acoustic drive level of approximately 7V_(pp) (FIG. 5B), the approximately 10 μm particles rapidly transportedto the axis of the capillary. The approximately 1 μm particles remainedrandomly distributed. Doubling the drive voltage to approximately 16V_(pp) resulted in efficient transport of both sized particles to thecentral axis of the capillary, see FIG. 5C. The bright line along theaxis of the cylinder was a result of the large, local increase influorescence due to the concentration of the approximately 1 μmfluorescent particles at that location.

Acoustic Focusing/Orientation Such as the Effects of Reflection Symmetryon Optical Scatter Parameters in an Acoustically Focused Flow Chamber

An embodiment of the present invention addresses angular scatteringassociated with particles that are aligned in the optical scatteringplane as a result of acoustic radiation pressure. The replacement ofhydrodynamic sheath flow with acoustically driven particle alignment ina flow cell preferably leads to improved light scatter data and yieldsnew parameters that are dependent upon particle geometry andorientation. In addition to the importance of particle orientation ofasymmetric biological particles (e.g. RBC's, sperm cells, bacteria) inflow cytometry analysis, complex geometries that are formed from thecoalescence of multiple microspheres or cells (e.g. ‘dumb bell’ shapesfrom agglutination of two particles) also benefit from particleorientation. Particle clusters are preferably more easily discriminatedby having their orientation fixed in the scattering plane. Orientingmicrosphere ‘doublets’ to yield repeatable and unique scatteringsignatures due to how they transit the scattering plane will provide ameans to isolate their contribution in optical scattering data foreither data rejection or data acceptance by utilizing the solution ofthe inverse scattering problem for contacting spheres. The applicationof acoustically oriented particles in a flow stream is also applicableto the field of imaging where viewing selected orientations of particlesis valuable in determining cellular morphology, localization of cellularconstituents, or other particle/cellular characteristics.

Enhanced Detection Capabilities Under Slow-Flow, Stop-Flow, andReverse-Flow Conditions in Acoustically Focused Flow Chambers

Another embodiment of the present invention further addresses theeffects of slow-flow, stop-flow, reverse-flow, and increased analysistimes in flow cytometry detection that result from replacing sheath flowwith acoustic particle alignment. In the first instance, the ability tostop and reverse the flow direction of the sample stream allows forparticles to be reanalyzed. The flow is stopped and/or reversed usingvarious stop flow and reverse flow devices, including but not limitedto, a pump or one or more valves. Peak spread (increased CV's) and datapoints that are outliers in the analysis plane are system dependentquantities that are a function of laser stability, quality of particlealignment, electronic noise, detector noise, robustness of the assay(on/off rates, etc.), etc. By analyzing a particle of significance morethan once, the data quality can be improved (especially in the case oftransient artifacts) and the statistical uncertainties in rare eventanalysis can be minimized.

Acoustic Field-Based Particle Size Selection for In-Line SamplePurification and Particle Isolation

While acoustic focusing is useful for particle or cell analysis byreplacing sheath flow in the detection system, yet another embodiment ofthe present invention extends the application of acoustic forces inacoustically driven capillaries to particle and/or cell separations forupstream, in-line sample conditioning in flow cytometry systems orgeneral sample preparation and purification. FIG. 6 illustratesutilizing the present invention to acoustically size fractionate (andconcentrate) samples by particle size and/or mechanical contrastrelative to the background medium in real-time at the inlet of a flowcytometer before the analysis stage. Direct fractionation based uponparticle size/mechanical properties alleviates the need for laborintensive sample preparation steps that include centrifugation andfiltering. For flow cytometry applications, this is useful in reducingthe background associated with cellular debris, proteins, and othermolecular components in whole blood assays, and in particular, nowashassays that include cellular lysis. A sample preparation including acellular debris rejection step prior to sample delivery into the flowcytometer can greatly reduce artifacts associated withscatter/fluorescence from the debris.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverall such modifications and equivalents. The entire disclosures of allreferences, applications, patents, and publications cited above and/orin the attachments, and of the corresponding application(s), are herebyincorporated by reference.

What is claimed:
 1. An apparatus that acoustically orients a particle ina flow stream comprising: a capillary configured for flowing a fluidcontaining the particle therein; an acoustic signal producing transducerconfigured to acoustically orient the particle; and a particle analyzer.2. The apparatus of claim 1, wherein said apparatus comprises a flowcytometer.
 3. The apparatus of claim 1, comprising a particle sorter. 4.The apparatus of claim 3 wherein said sorter separates the particlebased on size prior to analysis.
 5. The apparatus of claim 1, whereinsaid acoustic signal producing transducer aligns the particle in a polardirection about a flow axis.
 6. The apparatus of claim 1, wherein saidacoustic signal producing transducer aligns the particle in a directionof flow.
 7. The apparatus of claim 1, comprising a particlefractionator.
 8. An apparatus that separates particles by sizecomprising: a capillary configured for flowing a fluid containing theparticles therein; and a radial acoustic signal producing transducerconfigured to acoustically separate the particles in said capillary bysize by applying radial acoustic pressure to the particles.
 9. Theapparatus of claim 8, further comprising an imager.
 10. The apparatus ofclaim 8, wherein acoustically separating the particles in said capillarycomprises transporting larger particles to a substantially central axisof the capillary.
 11. The apparatus of claim 10, further comprising aflow cytometer, wherein the flow cytometer comprises an inlet centeredabout the substantially central axis of the capillary.
 12. The apparatusof claim 11, further comprising a particle sorter associated with theflow cytometer.
 13. The apparatus of claim 11, further comprising aparticle analyzer associated with the flow cytometer.
 14. The apparatusof claim 8, wherein acoustically separating the particles in saidcapillary by size is performed without gravity based separation.
 15. Theapparatus of claim 10, wherein the larger particles compriselymphocytes.
 16. An apparatus that separates particles by sizecomprising: a capillary configured for flowing a fluid containing theparticles therein, the particles comprising larger particles and smallerparticles; and an acoustic signal producing transducer configured toacoustically separate the particles in said capillary by size byapplying acoustic pressure to the particles.
 17. The apparatus of claim16, further comprising an imager.
 18. The apparatus of claim 16, whereinacoustically separating the particles in said capillary comprisestransporting the larger particles to a substantially central axis of thecapillary.
 19. The apparatus of claim 16, further comprising a flowcytometer, wherein the flow cytometer comprises an inlet centered aboutthe substantially central axis of the capillary.
 20. The apparatus ofclaim 19, further comprising a particle sorter associated with the flowcytometer.
 21. The apparatus of claim 19, further comprising a particleanalyzer associated with the flow cytometer.
 22. The apparatus of claim16, wherein acoustically separating the particles in said capillary bysize is performed without gravity based separation.
 23. The apparatus ofclaim 18, wherein the larger particles comprise lymphocytes.